Studies on Steroid Conjugates: Evaluation of Various Extraction Methods for Urinary Conjugated Corticosteroids

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Abstract
Various extraction methods for urinary conjugated corticosteroids, including several new techniques recently developed in this laboratory, were extensively studied and evaluated using the following criteria: 1) effectiveness in extracting conjugated Porter-Silber (P-S) chromogens and interfering “background” substances; 2) recoveries of pure corticosteroid conjugates: tetrahydrocortisone-3-glucuronide (THEG), cortisol-21-sulfate (FS) and tetrahydrocortisol-3,21-disulfate (THFdiS); 3) extraction of conjugated radioactive metabolites of cortisol-4-C14, which was administered iv as a tracer. Out of 22 techniques examined, 4 proved to be superior to the others. They gave the following results: 95–99% of conjugated P-S chromogens and 97–99% of the radioactive steroid metabolites were removed by one extraction either with one vol of n-butanol (method B), or with 4 vol of an ethanol-ether (1:3) mixture (method F), or with 5 vol of ethyl-acetate (method G), provided the aqueous phase was acidified to pH 1 and saturated with (NH4)2SO4 prior to the extraction. Techniques B and F gave the highest recovery of pure steroid glucuronide and sulfates. Method G removed only 88% of FS and no THFdiS, but gave equally as high a recovery of THEG as methods B and F, and extracted markedly less interfering “background” substances than the other techniques. The butanol-chloroform extraction procedure (method C) proved to be most advantageous for rapid routine estimation of urinary-hydroxycorticosteroids. The results of this study clearly demonstrated the usefulness of the extraction techniques in which the conjugates are forced into a relatively nonpolar solvent by acidification and saturation of the aqueous phase with ammonium sulfate. These “forced extraction” techniques were shown to have much greater selectivity than procedures using more polar solvents. The advantage of using solvent mixtures as extractants is also emphasized by the results of this study.